Osteonectin is a known protein, otherwise designated as SPARC (secreted protein, acidic and cysteine-rich), or as BM40 and 43 Kd. It is a secreted glycoprotein widely distributed in human and non-human animal tissues. It is associated with cell populations exhibiting high rates of turnover and remodelling [5,6]. The protein is secreted by different normal cells as a unique glycoprotein band of 40–44 kDa [5]. It has been shown that in addition to a reactive protein doublet of 40–45 kDa, osteonectin can appear in fresh melanoma samples and cell lines as a cleaved protein doublet of 34–35 kDa. Genes that encode osteonectin in several species, including the human, are also known. The gene encoding osteonectin is known to be highly conserved across a wide range of living organisms.
A diverse range of functions and effects has been associated with osteonectin. It has been found to interact with extracellular matrix components, growth factors, cytokines, and to regulate matrix metalloproteinase expression.
The literature of oncology includes numerous mentions of osteonectin and its synonyms. In particular, reports from different laboratories indicate that osteonectin over-expression was associated with neoplastic progression of different malignant tumours [8–12], including human melanoma. Porter et al [8] previously reported increased SPARC expression in ovary cancer cells, but on the other hand Mock et al have reported that SPARC expression is down-regulated in ovary cancer cells compared to normal cells [27], and that moreover the stable over-expression in ovary cancer cell lines of SPARC, secreted as a unique species of 43 kDa. reduced their growth capacity [27]. Molecular analysis of SPARC's role in cell growth thus has to take into account studies (including data of the present inventors) showing no effect on melanoma cell growth following SPARC down-regulation.
Osteonectin expression has been reported to be correlated with lung colonization by tumours. Increased expression has been reported in human breast cancer. Neoplastic progression of colorectal cancer has been reported as associated with over-expression of certain proteins including osteonectin.
Osteonectin has been reported as having effects on tumour cell adhesion and invasion. One view concerning cell-matrix interaction in tumour progression indicates that inhibition of tumour cell adhesion can result in less aggressive invasive behaviour. Adhesive proteins like fibronectin, vitronectin and laminin can regulate metastatic activity, invasive capacity and collagenase IV production of human and murine melanoma cells [13–16]. Osteonectin by contrast is considered to be a counteradhesive protein involved in matrix deposition and assembly due to its interaction with collagens types I–V [5,6,17], thrombospondin [5,18] and plasminogen [18] and the regulation of the expression levels of laminin [19], fibronectin [18,19], matrix metalloproteinases [7] and plasminogen activator inhibitor-1 [18].
Domains I and IV of SPARC, which can be specifically cleaved and released by different serine proteases [20], have been found responsible for SPARC binding to collagen IV and for disruption of focal cell adhesions [21,22].
Native SPARC and a specific peptide corresponding to domain II which exhibit sequence similarity with EGF-like domains have been shown to inhibit endothelial cell proliferation [23]. However, a second peptide corresponding to another region of domain II containing the Cu2+ binding sequence KKGHK [SEQ ID NO:2] which promotes cell growth and angiogenesis [24] stimulated endothelial cells and fibroblast proliferation [23].
Sage and colleagues reported that SPARC was able to inhibit DNA synthesis in endothelial cells, through a mechanism which did not involve changes in cell morphology [28]. Their evidence that peptides corresponding to domain II of SPARC can stimulate cell proliferation [23], suggests that the extracellular cleavage may have important consequences on the ability of SPARC to modulate cell growth. On the other hand, SPARC obtained from bovine bone, which efficiently modulated laminin and fibronectin secretion, was reported to have negligible effects on cell growth of different human malignant cell lines [19]. Stable over- or under-expression of SPARC in F9 cells, which correlated with altered morphologies in transfected cells, was reported not to affect cell growth [29]. In general, SPARC interaction with and regulation of the expression levels of matrix components seems to occur in the absence of any significant effect on cell growth [5,6]. Taken together, data from the literature and data of the present findings suggest that SPARC inhibition of cell growth can be related to specific cell types, probably independent of its modulation of cell matrix interaction.
Tumour cell rejection has been associated with a localized and massive inflammatory infiltrate of segmented neutrophils suggesting an increased availability of neutrophil trigger factors like IL-8 or the autocrine melanoma growth stimulating activity (MGSA/GRO) [30]. Matrix components like laminin were shown to modulate the availability of cytokines and chemokines [31] indicating that changes in matrix composition or in the interactions between tumour cells and matrix components may alter the immunological signals transduced to the immune system.
The concept of antisense strategies involving oligodeoxynucleotides and plasmid-derived RNA has previously been proposed as an attractive mode of anticancer gene therapy [32].
In certain cases the constitutive expression of antisense RNA coding for particular oncogenes and growth factors has been reported to be effective in the induction of reduced tumorigenicity or of in vitro malignant characteristics [33–36]. But, with few exceptions, complete suppression of tumour formation has not been achieved [32]. There thus remains a need for further materials and methods that can be used in the characterization and therapy of malignant disease and the modulation of tumour cell activity.
The present inventors have found that cellular downregulation of osteonectin in tumour cells can be associated with loss of tumorigenic effect, and that the effect can be transmitted from downregulated tumour cells to neighbouring untreated tumour cells. Downregulation of osteonectin can in particular cause a decrease in in-vitro adhesive and invasive capacity of tumour cells, and the present inventors consider that this is usefully connected with the loss of tumorigenic effect.
For example, it has been found that SPARC (osteonectin) inhibition in melanoma cells can suppress their tumorigenic capacity, providing the ability to use SPARC gene targeting for antisense therapy of human melanoma.
Evidence has been found, in particular, that SPARC (osteonectin) plays a key role in the tumorigenic capacity of human melanoma. It has been found for example that down-regulation of SPARC completely prevented tumour formation in nude mice. This effect was accompanied by a massive immune response in vivo and a strong decrease in the in vitro adhesive and invasive capacity of tumour cells. The present inventors have observed that with SPARC antisense-transfected cells no initial tumour formation was visible and tumour cell necrosis was observed very rapidly. This indicates that SPARC antisense-transfected cells were impaired in their ability to establish early tumour growth.